This invention relates to proteins with xylanase activity derived from bacteria, and in particular to xylanases which are free of any significant cellulase activity and which are active at high temperature and at neutral to alkaline pH. Xylanases having these characteristics are particularly useful in the bleaching of wood pulps, such as kraft pulps.
Enzymes are proteins present in all living cells, where apart from controlling metabolic processes, they break down good materials into simpler compounds. The enzymes are catalysts which speed up processes which would otherwise proceed very slowly, or not at all. Moreover, enzymes are very specific, breaking down only one type of compound.
Xylan is a polysaccharide found in most plant cell walls, consisting of D-xylose units linked by xcex2-1-4 glycosidic bonds. It occurs with another polysaccharide, cellulose and an amorphous binding polymer, lignin. Xylan forms a major component of plant hemicelluloses, and varies in the nature of substituents on the sugar groups, depending on the origin. For example, xylans derived from hardwoods typically consist of a backbone of O-acetyl-4-O-methylglucuronoxylan, in which about 10% of the xylose units carry 4-O-methylglucuronic acid side chains linked via xcex1-1,2 bonds, and 70% of the zyloge residues are acetylated at C-2 or C-3. In contrast, xylans derived from softwoods are usually arabino-4-O-methylglucuronoxylana in which over 10% of the xylose sub-units carry arabinofuranose residues linked via xcex1-1,3 bonds. Enzymes which are able to degrade xylan are called xylanses (endo-1,4-xcex2-D-xylanses; International enzyme nomenclature EC 3.2.1.8).
Commercial preparations of xylanase, often in combination with other cell wall degrading enzymes, have been used in the extraction or liquefaction of plant material. For example, in the food industry, the mashing process for the production of juices can be made to produce higher yields and better processing with the application of cell wall degrading enzymes, which include zylanase.
The primary source of cellulose for paper manufacture is wood, and may be either hardwood or softwood. The initial step in paper manufacture is the reduction of wood to the fibre state, which my be achieved by mechanical or chemical pulping methods. Chemical pulping involves the xe2x80x9ccookingxe2x80x9d of woodchips with chemical reagents in order to separate the cellulose fibres from the other wood components, and to break down the ligin and other extraneous compounds so that the cellulose in left in tact in its fibrous form. The most common process in the kraft or sulphate process, which can be applied to almost any timber species. The active ingredients are sodium hydroxide and sodium sulphide in a strongly alkaline solution.
During the kraft pulping process, xylan in the wood is initially dissolved in the pulping liquor, but with time, reprecipitates on to the resulting pulp. Wood lignin is modified d dissolved by the pulping liquors. However, about 10% of the liguin remains in the kraft pulp. To brighten the pulp, the lignin must be removed by bleaching chemicals, such as chlorine, which generate environmentally hazardous wastes.
More recently, commercial xylanase preparations have been used as an aid to the bleaching of kraft wood pulps. A program of cooperation between research institutes and the pulping industry has shown that treating the unbleached kraft pulp with xylanse results in a reduction in the amount of bleaching chemicals required to obtain a full brightness pulp. It is believed that xylanase acts as a bleaching aid (bleach booster) by releasing some trapped residual ligin within the pulp matrix and giving better access to bleaching chemicals. It is widely believed that xylanase breaks down reprecipitated xylan which forms a coating on the pulp, thus releasing trapped residual lignin from within the pulp matrix, and allowing better access of bleaching chemicals to this matrix. Thus xylanase acts as a bleaching aid or bleach booster.
In the kraft process, the pulp is typically handled at high temperatures and neutral to alkaline pH. Commercial xylanases typically have a temperature optimum of about 50xc2x0 C. and a pH optimum of about 5, and are thus subject to rapid denaturation under process conditions. Thus there is a need for xylanases which are able to act optimally on the kraft pulp without any requirement to adjust the temperature or pH. In order to be useful as a bleaching aid, the xylanase must also be free of any significant cellulase activity, since cellulase would cause an undesirable loss of cellulose fibre.
We have screened microorganisms newly isolated from a range of environments in order to identify those which produce high levels of xylanases with high temperature optima and which are active at neutral to alkaline pH. A previously unidentified bacterium isolated from white-rotted wood, produces such a xylanase in high yield and free of significant cellulase activity. Thus bacterium is a strain of Bacillus Subtilis which we have designated B230.
According to one aspect, the invention provides a bacterium, isolatable from wood compost, having the following characteristics:
A. Ability to grow at a temperature between 20xc2x0 and 45xc2x0;
B. Ability to grow in the pH range of 5 to 9.5;
C. Ability to grow on Luria-Bertani agar at 37xc2x0;
D. Ability to grow under solid state or submerged culture conditions; and
E. Constitutive production and/aor extracellular release of at least one protein with xylanase activity having an associated cellulase activity of less than 0.1, said at least one protein having a molecular weight of about 28 kD.
Preferably the bacterium is isolated such that a biologically pure culture exits.
Preferably xylanase production is enhanced by growth in the presence of xylan or of lignocellulose substrates, or degradation products, including xylose and xylitol, derived from such substrates.
More preferably the xylanase has at least one characteristic selected from the group consisting of activity at about pH between 4.5 and 9.5, a thermal activity range up to 70xc2x0 C., and high thermal stability up to 65xc2x0 C. Most preferably the xylanase produced by the bacterium is effective on both soluble and insoluble xylans.
In a particularly preferred embodiment, the bacterium has the characteristics of the bacterial isolate designated B230, as deposited under the provisions of the Budapest Treaty in the Australian Government Analytical Laboratories, PO Box 385, Pymble, New South Wales 2073, Australia, on Sep. 6, 1994, under Accession No. N94/41262, or a mutant or derivative thereof having the ability to produce a xylanase as described above. The term xe2x80x9cmutant or derivativexe2x80x9d thereof includes naturally occurring and artificially induced mutants which retain their ability to digest xylane. Production of such mutants or derivatives will be well known by those skilled in the art.
According to a second aspect, the invention provides a process for producing at least one protein with xylanase activity said process comprising cultivating a bacterium under conditions and for a time sufficient to produce said protein and collecting culture medium wherein said bacterium has the following characteristics;
A. Ability to grow at v temperature between 20 and 45xc2x0;
B. Ability to grow in the pH range of 5 to 9.5;
C. Ability to grow on Luria-Bertari agar at 37xc2x0;
D. Ability to grow under solid state or submerged culture conditions; and
E. Constitutive production and/or extracellular release of at least one protein with xylanse activity, said protein having an associated cellulase activity of  less than 0.1%.
Preferably the bacterium used is strain B320 or a mutant, variant or derivative thereof.
Preferably the bacterium is grown under optimal conditions for extracellular production of said at least one protein. Still more preferably the production of said it least one protein is induced by the addition of xylitol to the culture medium. Preferably xylitol is added in an amount of 0.01 to 2% of the culture medium which is preferably a broth.
According to a third aspect, the invention provides a protein with xylanase activity said protein having an associated cellulose activity of less than 0.1% and a molecular weight of about 28 kD as determined by SDS-PAGE. Preferably the protein has at least one characteristic selected from the group consisting of activity at about pH between 4.5 and 9.5, a thermal activity range up to 70xc2x0 C. and high thermal stability up to 65xc2x0 C. Preferably the protein is effective in digesting both soluble and insoluble xylans.
Preferably the protein with xylanase activity is isolatable from the bacterium described above. More preferably the protein is isolated from the bacterial strain B230.
Preferably the protein with xylanase activity is an isolated preparation meaning that it has undergone some purification away from other proteins and/or non-proteinatious material. The purity of the preparation may be represented as at least 40% protein with xylanase activity, preferably at least 60% protein, more preferably at least 75% protein with zylanase activity, still more preferably at least 80% protein with xylanase activity or greater, as determined by weight, activity, amino acid composition or similarity, antibody reactivity or any other convenient means.
According to a fourth aspect, the invention provides a composition comprising said protein with xylanase activity as an active ingredient together with an industrially acceptable stabilizer. The composition may be used as a bleaching aid or bleaching booster or in paper deinking. Those skilled in the art will be familiar with the types of industrially acceptable stabilisers which may be used such as glycerol, sorbitol or other polyalcohols.
The composition described above is for use in bleaching kraft pulp or deinking paper. Accordingly, in a fifth aspect the present invention provides a method of bleaching wood or paper pulp comprising administering a bleaching aid or bleaching booster effective amount of the composition to said pulp, for a time and under conditions sufficient to achieve the desired bleaching of the pulp.
The protein of the present invention may also be used in the preparation of animal feed and in preparation of dough for bread-making.
We have found that the bacterium B230, when grown under suitable fermentation conditions will produce xylanase which accumulates in the extracellular fermentation broth. The xylanase from such a broth has a thermal activity range from ambient up to 70xc2x0 C. and a useful pH range from 5 to 9, with optimal activity at pH 6-6.5. The xylanase has very high thermal stability, retaining 100% activity after 3 hrs and 90% activity after 22 hrs at 60xc2x0 C. Cellulase activity associated with the xlanase is minimal ( less than 0.1%).
The crude preparation may be used however partially purified may also be used.
While the following description refers to a single xylanase, our results indicate that there are in fact at least two different xylanase produced during fermentation of bacterium B230, and all xylanase produced by this organism are within the scope of the invention.
The invention will now be described by way of reference only to the following non-limiting examples, and to the figures in which: